RU2482133C2 - Chitosan composition - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a crossed-linked chitosan composition. The composition contains chitosan with a degree of deacetylation 30-75% and a cross-linking agent. Chitosan is randomly deacetylated. The molar ratio of the cross-linking agent to chitosan makes 0.2:1 or less at a number of functional groups in the cross-linking agent and a number of deacetylated amino groups in chitosan. The invention also represents chitosan hydrogel made of the specified composition, a method for preparing it and the versions of using it.

EFFECT: invention provides higher bioavailability of the active ingredient and ensures that hydrogel has storage-stability of the visco-elastic properties of a plastic solid.

15 cl, 1 dwg, 18 ex

Description

This invention relates to a chitosan composition, and especially to chitosan gels made from chitosan with a low degree of deacetylation and covalently crosslinked at a pH in the range of 6-10.

State of the art

The present invention relates to biocompatible polysaccharide gel compositions, and in particular chitosan compositions in vaccines, for drug delivery, tissue extension, cell cultivation, encapsulation of viable cells, cosmetic use, orthopedic use, use as biomaterials, wound healing devices, thickener and additives in the food industry, use as adhesives, lubricants, drilling fluids and auxiliary fluids. Gels are prepared using covalently crosslinked chitosan gels with a low degree of deacetylation. By selecting chitosans with certain degrees of deacetylation and using effective crosslinking conditions, gels with interesting and unexpected biological and physical properties can be obtained. They differ from other crosslinked chitosan hydrogels made from standard chitosan and using typical crosslinking protocols. The gels of the invention can be made which have very low toxicity, and they can be made to decompose quickly. Another unusual feature of these gels is that they do not precipitate when exposed to neutral and alkaline conditions. They also have rigidity, which allows additional mechanical processing, for example, into injectable, so-called "crushed gels", useful in a large number of applications.

A hydrogel can be defined as a colloidal gel in which water is a dispersion medium. Hydrogels are widely used in many fields and in several areas they have become multi-billion dollar industries. Typically, hydrogels are made from water-soluble polymers that have either been isolated from natural sources or obtained by synthesis or chemical modifications of natural polymers. Such polymers are selected according to their physical and biological properties and are used alone or in combinations depending on the desired product properties. Some polymers have physical properties that make them suitable for medical use, while others are used in the food industry, machining and manufacturing industries as lubricants, drilling fluids and auxiliary fluids, in cosmetics, biomaterial applications, and biotechnology as cell frames and so on. Different applications require different polymer properties and many technical applications are based on varieties of unprocessed mass available at low cost, while medical applications require highly purified varieties, often at high cost. Sometimes the physical properties of the polymer solution, such as viscosity, are the main parameter of interest, while in other applications the biological and toxicological properties for their functioning in the intended application become more significant.

Some polymers are used for filling purposes in tissue extension compositions where the gels are used either alone or together with hard granules. In other applications, for example for wound healing, drug delivery, vaccine carriers, other polymers with different properties are required to meet medical requirements. In general, properties such as viscosity, antimicrobial activity, adhesive ability or ability to absorb / retain water are all properties that should be taken into account. The ability to retain water and swell is usually of great importance in food applications where the polymer is used either as a thickener or as an solubility improver and stabilizer of other agents. There is a wide range of polymers found in medical products, both synthetic polymers and polymers of natural origin. In many applications, it is important that the polymers disintegrate and stand out without causing unwanted side effects. Although biodegradability is not always necessary, good biocompatibility is critical for preventing adverse reactions such as inflammation, immunological reactions, or substance rejection. Thus, it is not surprising that naturally-occurring non-toxic polysaccharides are used in medical products, since they have remarkable physical properties in combination with biological and medical properties of interest and are usually available with a high degree of purity and at a low price. Commonly used polysaccharides are, for example, cellulose, alginates, chitosan, hyaluronic acid, starch or derivatives thereof.

In medicine, gels and ointments are used, for example, for drug delivery, cosmetic purposes, or to create antimicrobial barriers to avoid infection. Hydrogels often have the desired solubility and biological properties and therefore are found in a large group of products. Gel-forming polysaccharides, such as hyaluronic acid, cellulose derivatives and the like, are becoming cost-effective industries. Hyaluronic acid is an example of a polymer that could be used on its own, as it spontaneously forms hydrogels when used in low concentrated aqueous solutions at physiological pH. Other polymers like cellulose cannot be used as such and must be chemically modified to obtain the desired properties. When producing hydrogels from polysaccharides, the usual protocol involves dissolving the polymer in an aqueous solution at low concentrations, often from 0.5 to 3% (w / w). When higher viscosities are required, this can be achieved either by adding more polymer to the solution, if solubility allows, or by crosslinking the polymers. Crosslinking gives polymers with a higher molecular weight and therefore higher viscosity. Crosslinking can be performed in various ways using covalent, ionic or hydrophobic strategies, and a large number of approaches are available. In the general case, when the product of such a crosslinking reaction is intended for medical use, it is desirable to ensure the level of crosslinking as low as possible, since there is a risk of incorporating immunological reactions against the linker, and this may also negatively affect biodegradation.

Immunology and allergies. The immune system can be divided into innate and adaptive immunity. Congenital or nonspecific immunity is the hereditary resistance exhibited by individuals who have not been immunized with an infection or vaccination. Adaptive or acquired immunity is a type of immunity in which there is an altered reactivity to an antigen that stimulates it and which creates an antigen-specific immunological memory. The immunity can be active, that is, a consequence of an acquired infection or vaccination, or it can be passive, that is, acquired by transferring antibodies. Passive antibody vaccination has several drawbacks: injection of foreign substances can elicit an immune response against injected antibodies. Monoclonal antibodies must be injected in large quantities, which makes this therapy very expensive. Treatment should be long to maintain their function. Active vaccination is most often preferred to induce antibody production and immunological memory. Most natural immunogens are proteins with a molecular weight of more than 5 kDa. Even immunogenic molecules may not create the right level of immunity. To increase the intensity of the immune response, immunogens are combined with adjuvants. Adjuvants are agents that enhance the immune response without forming unwanted antibodies against the adjuvant. If the immunogen is still unable to elicit an acceptable immune response, it can be conjugated to a carrier that is more immunogenic. Small molecules with a molecular weight in the range of 0.1 to 2 kDa are often too small to be recognized by the immune system, and are therefore inconvenient for use on their own in immunizations. One way around this is to bind them covalently with larger carrier molecules. The vaccination can be oral, nasal, subcutaneous, submucous, sublingual or intramuscular.

The recognition and destruction of foreign cells by T and NK cells is called cell-mediated immunity (TH1 immune response). Humoral immunity is associated with B cells (TH2 immune response). It has been reported that aluminum hydroxide selectively activates TH2 cells, while Freund's complete adjuvant activates TH1 cells. Chitosan has been proven to enhance both humoral and cell-mediated immune responses (Vaccine 3, pp. 379-384, 1985).

The innate immune system recognizes a wide range of pathogens without the need for preliminary testing. The main cells responsible for innate immunity, monocytes / macrophages and neutrophils, phagocytize microbial pathogens and initiate innate, inflammatory and acquired immune responses. Toll-like receptors (TLRs) are a family of type 1 transmembrane proteins involved in the recognition of a wide range of microorganisms. They play a major role in the innate immune system. TLRs are a type of image recognition receptor (PRR) and recognize molecules that are widely distributed in pathogens but different from host molecules, collectively called pathogen-associated molecular structures (RAMP). Macrophage receptors are also considered pattern-recognizing receptors. The macrophage mannose receptor recognizes hexoses with equatorially located hydroxyl groups on C ₃ and C ₄ carbon atoms in positions that enable recognition of mannose, fructose, N-acetylglucosamine and glucose (Curr. Opin. Immunol. 10, 50-55, 1998).

Allergy is a very common disorder, affecting approximately one fourth to one third of the population in developed countries, for example, more than 50 million Americans suffer from allergic diseases. The treatment strategy, which is generally the most commonly used at present, aims at the effector mechanisms of allergies, for example, by taking oral antihistamines or using local corticosteroids. Treatment with antihistamines and corticosteroids may be effective in alleviating the symptoms of allergies, but their use leads to the effects of the pharmaceutical product on the whole body, and they can cause unpleasant or even dangerous side effects. Allergen-specific immunotherapy is the only treatment used, the purpose of which is the underlying cause of the allergy, and which provides a lasting relief of the symptom. Thus, it can be considered the only curative treatment for an allergic disease. This treatment can be performed in the form of subcutaneous injections or sublingually. Carried out by injection of allergenic extracts subcutaneously, it has a documented effect, while the effectiveness of sublingual allergen-specific immunotherapy is less documented.

Allergic diseases, such as asthma and rhinitis, are caused by an inadequate immune response to other harmless environmental antigens, i.e. allergens. The most common form is immunoglobulin (Ig) E-mediated allergy, characterized by the presence of allergen-specific IgE. Currently, there are two main strategies for the treatment of IgE-mediated allergies, pharmacological therapy and allergen-specific immunotherapy. Pharmacological treatment includes treatment with topical corticosteroids, especially in the case of allergic asthma and eczema. However, 10-20% of patients with allergic asthma do not respond to steroid treatment. Other common antiallergic drugs target the effector mechanisms of IgE-mediated allergies, such as antihistamines, antileukotrienes, and chromones. The only radical therapy for IgE-mediated allergy, that is, the only treatment that provides lasting relief of symptoms, is allergen-specific immunotherapy (ASIT). It has been proven that, unlike pharmaceutical treatments, ASIT also reduces airway inflammation and protects against the development of chronic asthma (J Allergy Clin Immunol. 1998 102 (4 Pt 1), 558-62). Treatment is based on repeated administration of an allergen to induce allergen-specific immunity. Currently, ASIT widely uses allergenic extracts obtained from natural sources and adsorbed on aluminum hydroxide (alum). Alum delays the release of the allergen and acts as an adjuvant. However, there are some disadvantages associated with the use of allergenic extracts and alum. Numerous injections of low doses of allergen are required for 3-5 years. To solve such problems as the induction of new sensitization and harmful side effects with extracts, recombinant allergens have been proposed for use in ASIT (Adv Immunol. 2004; 82: 105-53, Nat Rev Immunol. 2006 Oct; 6 (10): 761-71) . Recombinant allergens can be modified in various ways to achieve safer and more effective protocols for ASIT. Examples of such new strategies are the creation of so-called hypoallergens, i.e. allergens with reduced ability to bind IgE but retaining T-cell activity, vaccination with peptides derived from the allergen, or binding of allergens to immunomodulating agents such as immunostimulatory oligonucleotides containing CpG motifs (Nat Rev Immunol. 2006 Oct; 6 (10): pp. 761-71, Curr Opin Immunol. 2002 Dec; 14 (6): pp. 718-27). Alum is known to cause granuloma at the injection site and primarily stimulate Th2 responses. Thus, there is a need for alternative adjuvants for ASIT.

Adjuvants are substances that enhance the ability of an antigen to elicit an immune response. Although large-scale efforts are being made to develop new adjuvants for human vaccines, aluminum hydroxide is still the only widely used adjuvant. It has been proven that aluminum adjuvants can cause neuronal death. The development of new adjuvants is desirable in order to maximize the effectiveness of new vaccines. An ideal adjuvant should provide long-term expression of functionally active antibodies, induce cell-mediated immunity, and enhance the production of memory T and B lymphocytes with highly specific immunoreactivity against antigen. It should provide both immediate protection and protection against future antigen infections. In addition, it must be biodegradable, non-toxic and not induce an immune response directed against the adjuvant itself.

Vaccinations should provide a lasting effect, rapid antibody production and high antibody titers.

The use of chitin and chitosan as an adjuvant is mentioned in US Pat. Nos. 4,372,883 and 4,814,169. The use of chitosan in vaccines in the form of solutions, dispersions, powders or microspheres has been described in US Pat. Nos. 5,554,388, 5,744,166 and WO 98/42374. Chitosan crosslinking switches the immune response from TH2 to a mixed TH1 / TH2 response. The use of chitosan solutions mixed with antigens for immunization shows that chitosan has the same strength as Freund's incomplete adjuvant and is superior to aluminum hydroxide (Vaccine 11, 2085-2094, 2007).

Drug delivery. Drug delivery is a very significant area of research, and a lot of money is being spent today on finding new and improved compositions that deliver pharmaceutical active ingredients such as low molecular weight drugs, genes, and vaccines, while minimizing unwanted side effects. Earlier drugs are new in new and improved formulations.

The properties of chitosan, physical and biological, have made it most suitable for the delivery of pharmaceutically active components and as a delivery vehicle, for example, for vaccines, gene fragments, and micro-RNAs. Useful and important features of chitosan are that it combines with any living tissue, has mucoadhesive properties, decomposes and opens up tight joints between cells. By using these properties, drug delivery through the mucous membrane can be significantly improved. Compositions of drugs based on technology using chitosan today are developed for various purposes, for example, as carriers of vaccines, hydrogels, drug-releasing drugs, membranes, mesh material and so on. Chitosan has been shown to be useful, for example, in drug delivery to the colon (H. Tozaki, et. Al, J. Pharm, ScL, 86, pp. 1016-1021, 1997) and intranasal insulin delivery (US 5744-166 ) In addition, chitosan was used as a carrier in gene delivery (MacLaughlin, et. Al, J. Controlled Release, 56, pp. 259-272, 1998).

Some compositions are designed to provide a sustained release over time, while release from others is faster. When using chitosan hydrogels, cross-linking was found to be preferred since gels without cross-linking agents tend to dissolve. Another advantage of using crosslinking is that the rate of release from the gel can be varied by using different degrees of crosslinking. Chitosan can be used to develop new compositions for, for example, oral, cutaneous, subcutaneous, buccal, sublingual, nasal, rectal, vaginal and intramuscular administration.

A large number of drugs that are administered in unmodified form by generally accepted systemic routes fail to reach target organs in effective concentration, or they are not effective for a long period of time due to the ease of metabolism. Using Drug Delivery Systems (DDS) can overcome these problems.

Anticancer drugs often have a short plasma half-life and / or noticeable side effects. Focal administration, that is, local delivery of the drug to the cancer area via implantation / injection of DDS containing a chemotherapeutic agent, may be an approach to reducing these problems. Compared to systemic administration, the magnitude of side effects will decrease, and the overall effect of the drug will increase.

When developing DDS for focal cancer therapy, some technical factors should be taken into account, namely biocompatibility, biodegradability (importance depends on the disease, area of application and number of administrations), sterility / sterilization, compatibility with drugs and pharmaceutical excipients, ease of administration (preferably with a syringe), flexibility with respect to dosing, drug loading, dose setting, ability to control the rate of drug release tools and tolerated by the patient, as well as consideration of procedural difficulties, CoG (cost of goods) and IPR.

By injecting DDS with the drug, a large amount of the loaded drug is localized to the tumor area, thereby improving cancer therapy and reducing the harmful non-specific side effects of chemotherapy.

Tissue extension. Tissue extension can be used for medical and cosmetic purposes. Medical use is, for example, tissue extension in order to achieve improved function of this tissue. Examples of tissues that can be enhanced by injection of volume-enhancing agents are the vocal cords, esophagus, urethra, or rectum. In the field of cosmetic surgery, soft tissue extension can be used to correct defects such as scars and wrinkles and to enlarge, for example, lips or breasts. To build or restore soft tissue, many different substances, both biodegradable and biodegradable, were used. Examples of substances used for long-term soft tissue extension are silicone, Gore-Tex and ePTFE (porous polytetrafluoroethylene). Examples of biodegradable substances are collagen, autogenous fat, crosslinked hyaluronic acid, and synthetic polymers.

Silicone is one of the most commonly used substances for the long-term build-up of soft tissue. Adverse reactions to liquid injectable silicone include granulomatous reactions, inflammatory reactions, and displacement. Such reactions can occur years after the initial treatment. In addition, since the injectable silicone is a long-term filler, the above complications can become a serious problem, since the substance is not metabolized, and the reaction may persist despite treatment.

Collagen is one of the most commonly used injectable substances, both for cosmetic applications and as a bulking agent, such as for urinary incontinence. Collagen, however, has some disadvantages. It breaks up quickly, and approximately 3% of the population shows delayed-type hypersensitivity reactions, which makes it necessary to perform allergic tests for the time before the injection. In addition, bovine collagen can transmit viral diseases.

Autogenic fat injections are well known. These substances also have disadvantages. Fat injected into facial wrinkles and creases caused vision loss and embolism in some patients. In addition, autogenic fat is easily absorbed by the body.

Crosslinked hyaluronic acid products are used both for cosmetic treatments and as volume-increasing agents in the treatment of, for example, urinary incontinence (UI) and vesicoureureral reflux (VUR).

A general approach in creating volume-increasing agents is to use spheres of a biodegradable substance distributed in a biodegradable carrier. Examples include carbon-coated beads in beta-glucan gel, hydroxyapatite spheres in carboxymethyl cellulose, polytetrafluoroethylene particles, and lactic and glycolic acid copolymer microspheres (PLGA). One danger when injecting particles is the possible migration of particles to distant organs such as the brain and lungs.

Existing substances are not optimal, and there is a constant search for new substances for use in tissue extension, substances injected through thin needles, biocompatible, non-toxic and with a suitable retention time in the tissue.

Chitosan gels for soft tissue extension have been described (WO 97/04012, EP 1333869).

Chitosan gels have also been used in cell culture and for incorporation of viable cells to be used, for example, in tissue engineering of cartilage, as described, for example, in Biomaterials. 2000; 21 (21): 2165-61, J Biomed Mater Res A. 2007; 83 (2): 521-9, and Biochimie. 2006; 88 (5): 551-64.

In cosmetics, chitosan was used, for example, in skin creams (US 20060210513, US 20040043963) and to reduce skin irritation caused by shaving (US 6719961).

Chitosan can also be used as a lubricant (Nature. 2003, 425: 163-165). The use of chitosan as a thickener has been described, for example, in Environ Sci Technol. (2002) 36 (16): 3446-54; and Nanotechnology (2006) 17 3718-3723. In addition, it was used as an adhesive (Biomacromolecules. 1 (2): 252-8 (2000) and Fertil Steril, 84, 75-81 (2005)) and as a food additive (US 5098733, US 5976550, US 6238720 and US 6428806).

In addition to medical use, viscoelastic chitosan hydrogels can be used as pseudoplastic, shear thinning chitosan-containing fluids, and a method for improving the thermal stability of such fluids is described, for example, in US Pat. No. 6,258,755.

Chitin is the most common polysaccharide on earth following cellulose. It is found in rigid structures and durable substances in which it has the function of a stiffener. Together with calcium salts, some proteins and lipids, it forms the outer skeletons of marine organisms like crustaceans and arthropods. It is also found in the cell walls of certain bacteria and sponges, and it forms hard shells and wings of insects. On an industrial scale, chitin is isolated from shellfish, which is a waste of the fishing industry. Chitosan is a linear polysaccharide formed by residues of 1,4-beta-linked D-glucosamine and N-acetyl-D-glucosamine. Chitin is not inherently water soluble, which greatly limits its use. However, the treatment of chitin with a strong alkali gives a partially deacetylated and water-soluble chitosan derivative, which can be processed into a number of different physical forms, for example, films, spongy materials, beads, hydrogels, and membranes. Chitosans in their basic form, and in particular high molecular weight chitosans, and / or with high degrees of N-deacetylation, are practically insoluble in water, but their salt with monobasic acids is usually water-soluble. The average pKa of glucosamine residues is about 6.8, and the polymer forms water-soluble salts, for example with HCl, acetic acid and glycolic acid. The solubility of chitosan depends on several factors, such as internal, such as, for example, chain length, degree of deacetylation, distribution of acetyl groups in the chains, as well as external conditions such as ionic strength, pH, temperature, and solvent. From the literature it is known that the degree of acetylation of approximately 50% is optimal for solubility. In the preparation of gels and aqueous solutions in an acidic medium, there is a practical limitation imposed by the solubility of a particular chitosan, which depends on its molecular weight and its degree of N-deacetylation. However, the amount of chitosan in the aqueous medium is usually in the range of 1-10% or 1-5% of the mass. in terms of the mass of the liquid medium, with an amount approaching the higher end of the interval, if low molecular weight chitosans are used (Carbohydr. Polym. 25, 65-70, 1994).

The characteristic properties of chitosan, which makes it biodegradable, non-toxic and antimicrobial, together with its cationic and hydrophilic nature, make it attractive in pharmaceutical compositions. However, its poor solubility under physiological conditions limited its practical use. Scientists have overcome this lack of solubility by making chemically modified chitosan derivatives with exceptional solubility properties at physiological pH, such as sulfated chitosan, N-carboxymethylchitosan, O-carboxymethylchitosan and N, O-carboxymethylchitosan (Int J Biol Macromol. (4), 177-80 1994, Carbohydr Res. 302 (1-2): 7-12, 1997).

The introduction of chemical substituents into chitosan results in altered biological properties, for example, an altered decomposition rate and the risk of introducing groups that will have a negative effect on biocompatibility and toxicity. This problem is addressed by US 6344488, where glycerophosphate is used as a solubility enhancer, and thus provides chitosan hydrogels at physiological pH without modifying the structure of chitosan.

Chitosan solutions can be crosslinked under acidic conditions, usually at a pH suitable for the formation of a Schiff base (pH 4-5), with the formation of hydrogels. A very large number of different crosslinking agents with different structures and reactivities were used. Some crosslinking agents have been used to form gels from liquid chitosan, for example glycosaminoglycans such as hyaluronic acid and chondroitin sulfate (Ann, Pharm. Fr. 58 47-53, 2000), glutaraldehyde 30 (Ind. Eng. Chem. Res. 36: 3631-3638, 1997), glyoxal (US 5489401), diethyl squarate (Macromolecules 31: 1695-1601, 1998), diepoxides such as diglycidyl ether (US 5770712), tripolyphosphate (J Appl Polym Sci 74: 1093-1107, 1999), genipin (J Polym Sci A: Polym Chem 38: 2804- 2814, 2000, Biomaterials. 23: 181-191, 2002), formaldehyde (J. Polym. Sci. Part A: Polym. Chem. 38, 474, 2000, Bull. Mater. Sci., 29, 233-238, 2006 ) If the desired product is a hydrogel, it is necessary that chitosan and its derivative remain in solution and avoid precipitation. Attempts to adjust the pH of crosslinked chitosan hydrogels to physiologically acceptable levels lead to precipitation and insoluble substances of limited use. It is desirable to keep the degree of crosslinking as low as possible, both for toxicological reasons and because the high degree of crosslinking can completely change the behavior of chitosan (Eur J Pharm Biopharm. 2004, 57 (1): 19-34. Review).

A special group of hydrogels are viscoelastic gels that are viscous and at the same time exhibit elastic properties. A viscoelastic gel is deformed and flows under the action of an applied shear stress, but if the stress is removed, the fluid slowly recovers from some deformation. It is used, for example, in ophthalmology, tissue extension and cosmetic surgery. The viscoelasticity of the gels allows machining, which includes the production of crushed gels. Viscoelastic hyaluronic acid gels are used, for example, in eye surgery, wrinkle filling, or in the treatment of urinary incontinence.

Chitosan, a natural polyelectrolyte. The three-dimensional orientation of the polyelectrolyte in an aqueous medium will depend, for example, on its natural / chemical composition, size, concentration and charge density, i.e. the number of charges and the distance between its charged groups. The spatial interactions of any polyelectrolyte in a solution are controlled by enthalpy, and the molecule tends to assume a low energy state in which it is most stable. This way of minimizing energy involves different types of interactions, or intramolecular (inside the same molecule) or intermolecular (between molecules). Examples of intramolecular interactions are hydrogen bonds, hydrophobic interactions, and interactions between charged groups in a polymer. Typical intermolecular interactions are solvent interactions and interactions with other molecules. Regardless of the type of interaction involved, the driving force behind these interactions is to find energetically favorable polyelectrolyte conformations.

If the polyelectrolyte contains charged groups, with the same type of charge, for example positive, these groups will repel each other. In order to reduce their internal energy, the polyelectrolyte molecule will try to carry its internal charges as far as possible, which will lead to an extended polymer chain. Such stretched polymers will not only be more "bulk", they will also have a relatively high state of energy, hidden in the strained bonds between the atoms.

On the other hand, if the polyelectrolyte contains charges of opposite signs, they will attract each other and form internal salt bridges, which will lead to a different three-dimensional orientation of the polymer, that is, different parts of the polymer will be brought closer to each other. Nonionic interactions do not occur in the polymer without any charges, and therefore its three-dimensional orientation will depend on its ability to form stabilizing hydrogen bonds and hydrophobic interactions within the molecule and with surrounding molecules and the environment. Unlike polyelectrolytes, uncharged polymers that do not contain any high-energy repulsive forces form a kind of “statistical spiral”, where their internal energy is minimized and their relative energy content is lower than that of polyelectrolytes.

In the physical sense, ionic interactions (charges) are stronger and involve more energy than other interactions such as hydrogen bonds, Van der Waals forces and hydrophobic interactions. The relative influence of the former on the molecular orientation is thus significant and in many cases obscures the influence of other types of forces involved.

The chitosan polymer with its mixture of N-acetylglucosamine and glucosamine residues could theoretically be a neutral polymer, but in most practically and biologically relevant situations it is protonated, since the pKa for glucosamines in chitosan is approximately 6.8. However, unlike polyelectrolytes bearing constantly charged groups, the charge density of the chitosan polymer can vary and be directly dependent on the pH of the aqueous solution. In practice, most commercially available and unmodified chitosans are insoluble in aqueous solutions, where the pH is higher than about 6, and above this pH they will precipitate from the aqueous solution. The deposition is energetically controlled, since the chitosan molecule requires a large number of charges on its molecular basis to form an energetically favorable state of solvation. If this cannot be achieved; molecules will precipitate from solution and form more stable precipitates. In the sediment, chitosan chains come together, which makes it possible to optimize energy using molecular interactions between and within chitosan molecules.

In order to increase the viscosity of the chitosan solution, chemical crosslinking can be used. In such a reaction, chitosan chains combine to form larger network-like aggregates. During this interaction, the viscosity gradually increases, and the solution becomes more gel-like in structure. There are a large number of crosslinking methods described for chitosan in solution, and the common one is that chitosan is dissolved in the acidified aqueous phase and the crosslinking reaction occurs at a low pH, usually 4-5. Used low pH means that the chitosan chains are in their protonated form, and they are thus in a "stretched" form when crosslinked. The resulting crosslinked gel, thus, from a technical point of view, is a macrostructure of protonated and stretched chitosan chains. If such a macronet is transferred to neutral or alkaline conditions, it gradually loses its charges, contracts, and ultimately precipitates. This is somewhat expected, since when standard chitosans (deacetylation degree between approximately 80-95%) are adjusted to a pH above 6, they precipitate. Crosslinking in itself creates even larger electrolytic structures, for which stabilization in an aqueous solution from an energy point of view will be even more required. This is due to the fact that the positive charges have become closer to each other at the contact points between the chains, and thus it is even more difficult to stabilize with the help of solvating water molecules. Thus, they are even more prone to precipitation than single chains, when the conditions in the solution change in a less energetically optimal way, for example, the pH rises. As a result of cross-linking, the macrogel structure is fixed in a stretched and energetically unfavorable state, which physically does not allow rearrangement in spirals and other conformations, which can contribute to energetically more preferred conformations, leading to higher stability of the system.

Precipitation of chitosan gels formed under acidic conditions can easily be confirmed experimentally by exposing a piece of such a crosslinked chitosan gel to a pH above seven or even higher values, i.e., pH 7-14. Immediately upon placing such a piece in a buffer with a higher pH, the surface of the piece becomes whitish due to the thin layer of sediment, and as diffusion continues, the piece becomes more and more whitish until it is completely precipitated.

However, unexpectedly, the inventors found that such a compression of the macrostructure of a crosslinked chitosan gel can be avoided by using low deacetylated chitosan with specific degrees of deacetylation and crosslinking of the chitosan chains in an energetically less stressed conformation. The gels obtained according to this method can be treated with 1 M sodium hydroxide without precipitation. A corresponding non-crosslinked gel will precipitate upon treatment with 1 M NaOH.

Using the higher solubility of these particular chitosans, the pH during the crosslinking reaction can be adjusted to a significantly higher value. The benefits of this are numerous. Firstly, the protonation of the chitosan chain becomes low, and the chitosan polymer is almost neutral at a pH above eight, allowing a less strained and more statistical helix, similar to a network, to form in solution. If the chitosan is crosslinked in this state, the resulting gel structure will be created by separate chitosan chains with higher flexibility, which will make them more readily reorganizable into energetically more preferred macrostructures under changing conditions. Secondly, the ability to use a higher pH value is beneficial from the point of view of significantly increased reactivity of amino groups in glucosamine residues. This makes the compounds more effective and makes it possible to use lower concentrations of crosslinking agents to achieve a certain degree of crosslinking. Another advantage is that adverse reactions are kept low. Such crosslinked gels have some advantages over chitosan gels obtained at low pH and from chitosans of standard grades (degree of deacetylation of 80-95%). The fact that they do not precipitate under physiological conditions means that they are more accessible to destructive enzymes, which leads to rapid degradation of gels, as well as other properties that are described in the present description of the invention.

Summary of the invention

Thus, the present invention provides a crosslinkable chitosan composition containing chitosan with a degree of deacetylation of 30 to 75%, where chitosan is deacetylated randomly, and a crosslinking agent, where the molar ratio of crosslinking agent to chitosan is 0.2: 1 or less, in terms of by the number of functional groups in the crosslinking agent and the number of available amino groups in chitosan.

The present invention provides the formation of a crosslinked, viscoelastic chitosan hydrogel at physiological pH without the use of other solubility enhancers, for example glycerophosphate.

One objective of the present invention is to provide a viscoelastic gel from chitosan that can be delivered at physiological pH without precipitation.

One objective of the invention is to provide a viscoelastic gel that has been given physical strength through chemical crosslinking, which allows further processing into smaller, individually separated gel fragments, for example, crushed gel.

Another object of the invention is to provide a viscoelastic chitosan hydrogel that can be delivered by means of a thin syringe needle, usually such needles that are used for injection, for example for vaccination.

Another object of the invention is to provide a crushed viscoelastic chitosan hydrogel that exhibits a large surface area and thus becomes readily available to enzymes and penetrating cells when used in vivo.

Another object of the invention is to provide a rapidly degradable viscoelastic chitosan hydrogel.

Another object of the invention is the provision of a viscoelastic chitosan hydrogel with low toxicity and the minimization or complete prevention of undesirable immunological or toxicological reactions when using low concentrations of crosslinking agents, preferably agents with low toxicity or non-toxic agents.

Another object of the invention is the proposal of a viscoelastic chitosan hydrogel, which provides the ability to include antigens and immunogens during its manufacture, either covalently or non-covalently.

Another object of the invention is to provide a viscoelastic chitosan hydrogel having characteristic adjuvant properties.

Another object of the invention is to provide a biodegradable, viscoelastic chitosan hydrogel for immunological use.

Another object of the invention is to provide a carrier for the delivery of antigens and immunogens intended for immunization. The present invention provides an immunological agent comprising a chitosan hydrogel, as described herein, and an antigen, where the antigen is possibly covalently linked to chitosan.

Another object of the invention is the proposal of a viscoelastic chitosan hydrogel, which provides the possibility of covalent incorporation of molecules leading to an immunological response. Said molecules can be either low or high molecular weight molecules, for example small molecules such as peptides, lipids, steroids and antibiotics, or large molecules such as proteins, gene fragments, micro-RNAs, carbohydrate polymers and synthetic polymers.

Another object of the invention is to provide a viscoelastic chitosan hydrogel containing more than one immunogenic substance.

Such a hydrogel can be made containing two or more antigenic molecules or mixtures of low and / or high molecular weight antigens.

Another object of the invention is the proposal of a viscoelastic chitosan hydrogel, which allows the inclusion of other agents, either covalently or non-covalently, for example, preservatives and other polymeric substances.

Another object of the invention is the proposal of a viscoelastic chitosan hydrogel, the releasing properties of which can be further changed by coating with anionic polymers. Using sequentially cationic and anionic polymers, it is possible to construct multilayer coated viscoelastic gel structures.

Another object of the invention is the provision of a viscoelastic chitosan hydrogel for use in adjuvant immunotherapy to nonspecifically enhance the immune response.

Another object of the invention is to propose a composition that provides a delayed release of the included bioactive agents or antigens (eg, allergens) by introducing them into a viscoelastic chitosan hydrogel.

Another object of the invention is the provision of a viscoelastic chitosan hydrogel for use in tissue extension, either alone or together with hard granules.

Another object of the invention is to provide a viscoelastic chitosan hydrogel for the treatment of, for example, urinary incontinence (UI) and vesicoureteral reflux (VUR).

Another object of the invention is to provide a viscoelastic chitosan hydrogel for use as a bulking agent.

Another object of the invention is the provision of a viscoelastic chitosan hydrogel as a framework for cell culture.

Another object of the invention is to provide a viscoelastic chitosan hydrogel for use in providing live cells of the host body.

Another object of the invention is to provide a viscoelastic chitosan hydrogel for oral, nasal, subcutaneous, submucous, sublingual, corneal, rectal, vaginal or intramuscular drug delivery. The present invention provides a pharmaceutical composition comprising a chitosan hydrogel, as described herein, and a pharmaceutically active ingredient.

Another object of the invention is the proposal of a viscoelastic chitosan hydrogel, which provides the ability to include and release drugs, for example, for focal cancer therapy, as described above in this description of the invention.

Another object of the invention is the proposal of a viscoelastic chitosan hydrogel, which provides the ability to include drugs in the manufacturing process, either covalently or non-covalently.

Another object of the invention is to provide a viscoelastic chitosan hydrogel for use as a device for treating wounds.

Another object of the invention is the proposal of a viscoelastic chitosan hydrogel for viscometer surgery.

Another object of the invention is to provide a viscoelastic chitosan hydrogel for cosmetic use.

Another object of the invention is to provide a viscoelastic chitosan hydrogel for use as a lubricant.

Another object of the invention is to provide a viscoelastic chitosan hydrogel for use as an adhesive.

Another object of the invention is to provide a viscoelastic chitosan hydrogel for use as a drilling fluid and auxiliary fluid.

The following is a description of the present invention with reference to the accompanying graphic material, in which Fig. 1 shows histological sections of injection sites of mice 24 parts after subcutaneous injection with the viscoelastic chitosan gel of the present invention and a comparative gel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to hydrogels made from chitosan, for example, intended for use in the treatment of humans or in veterinary medicine. More specifically, the present invention is directed to chitosan hydrogels with viscoelastic properties for use as described above. Thus, according to the invention, there is provided a composition comprising chitosan or mixtures of chitosans with different degrees of deacetylation, which do not precipitate under physiological conditions and at the same time exhibit a large surface area, thereby facilitating additional biological treatment. Compared to the prior art chitosan solution, the surface area of the viscoelastic hydrogel of the present invention is several orders of magnitude larger. This property of the viscoelastic hydrogel of the present invention causes faster cell infiltration and a faster immune response when the hydrogel is injected subcutaneously in mice compared to prior art chitosan solutions (Vaccine 11, 2085-2094, 2007). This opens up the possibility of using lower doses of the immunogen.

This is achieved by obtaining a crosslinkable chitosan composition containing chitosan with a degree of deacetylation of 30-75%, where chitosan is deacetylated randomly, and a crosslinking agent, where the molar ratio of crosslinking agent to chitosan is 0.2: 1 or less, based on the number of functional groups in the crosslinking agent and the number of available amino groups in chitosan. The chitosan hydrogel of the present invention is obtained by preparing this crosslinkable chitosan composition in an aqueous solution, crosslinking the composition and isolating the resulting chitosan hydrogel. The present invention also provides a hydrogel obtained by this method.

The solubility of chitosan depends on several factors, such as internal, such as, for example, chain length, degree of deacetylation, distribution of acetyl groups within the chains, as well as external conditions, such as ionic strength, pH, temperature, and solvent. Numerous attempts have been made to make physiological chitosan solutions and gels for medical use, but most often with unsatisfactory results. Most commercial chitosans have a deacetylation degree of more than 80% and for the manufacture of solutions and gels a low pH is required to dissolve the polymers, acidic solutions of acetic acid or hydrochloric acid are usually used. Attempts to raise the pH of such solutions result in precipitation of the chitosan polymer when the pH exceeds about 6. This problem can be circumvented by using numerous water-soluble derivatives of chitosan or by using additives like glycerophosphate. The inventors have found that chitosan with a low degree of deacetylation, for example 50%, can be used to produce hydrogels under physiological conditions and that chitosan concentrations can be prepared up to several percent. Commercially available chitosans with low degrees of deacetylation are limited, but the manufacture of such chitosans is described in the literature. One way to make chitosans with a low degree of deacetylation is to reacylate chitosan under acidic conditions and then deacetylate. Another approach is to start deacetylation with chitin, which is taken in solution under very alkaline and cold conditions. Chitosans with a low degree of deacetylation, for example 50%, are not only more soluble than chitosans with higher degrees of deacetylation, they are also faster and easier to cleave with hydrolytic enzymes, which require N-acetyl groups to recognize cleavage sites. A less obvious, but also important advantage of bringing chitosan gels and solutions to physiological pH levels is the concomitant increase in the reactivity of glucosamine residues in the polymer chain. In some circumstances, this can be used to suppress undesirable side reactions and to minimize the effect of possible crosslinking agents, since their concentration can be kept lower due to the increased reactivity of the amino groups, which leads to a more effective combination. An illustrative example of this is crosslinking of chitosan using a large excess of diethyl squarate under acidic conditions. In this reaction, which is carried out at a pH of about 4.75, almost 50 percent of the added reagent is hydrolyzed at one of the two reaction sites to give a chitosan chain substituted with squaric acid, which no longer crosslinks. When such an interaction is performed at a pH above 7, the reactivity increases by several orders of magnitude, and competing adverse reactions are suppressed, which leads to pure and effective interactions with a minimum of reagent to obtain the desired crosslinked network. This is most important when chitosan gel structures are intended for immunological use. There are several reasons for this. Firstly, there is always a risk of unwanted immunological reactions against the linker, secondly, there is a risk of altered decomposition kinetics, and thirdly, crosslinking linkers themselves usually have high reactivity and, if not completely consumed, can cause toxic side reactions Thus, it is important to keep the level of such agents as low as possible. The inventors prefer to use crosslinking agents with low toxicity and to use crosslinking agents that do not eliminate the groups that must be removed before the biological use of the viscoelastic hydrogel. Dibutyl Squarate is approved by the FDA for use as a local immunomodulator and is not mutagenic in Ames analysis. Another common group of crosslinking agents are reactive particles based on epoxy chemistry. Diglycidyl ethers are often used for various crosslinking reactions, where they interact with carboxylic acids, alkylates and amines. If diglycidyl ethers or a similar derivative were used to cross-link chitosan, rather high ratios of the cross-linker to the amino group of chitosan were used. This is illustrated in Journal of Biomedical Materials Research Part A, 2005, 75A, 3, 742-753 Eur. J. Pharm. Biopharm. 2004, 57: 19-34, US 5770712, WO 02/40070. The inventors have tried other crosslinking methods according to the literature. Glutaraldehyde crosslinking led, for example, to colored hydrogels, which precipitated under the action of an alkaline medium. The viscoelastic hydrogel according to the invention can be easily coated, for example, with anionic polymers in order to change the rate of biological treatment. If the viscoelastic hydrogel of the invention is dialyzed against an alkaline medium, the surface of the gel remains transparent, while dialysis of the gel results in a chitosan precipitate that is visible as a cloudy gel surface. This is a significant difference in precipitation between unstitched and crosslinked hydrogel. Unexpectedly, these gels could be treated with 1 M NaOH without precipitation.

The viscoelastic gel structure is affected by the concentration of the chitosan solution and the amount of crosslinking agent used. The inventors prefer to have higher concentrations of chitosan and lower concentrations of a crosslinking agent to obtain a gel of the required quality. The crosslinking molecule in this context has at least two reactive sites, which are electrophiles designed to easily interact with amines in neutral or slightly alkaline conditions. If the crosslinking agent has two reactive sites, it is bifunctional and thus can interact with two amino groups, for example two glucosamine units in different chitosan chains. There are a number of commercially available cross-linking agents of this nature and sometimes the distances between the reactive groups have been increased with the help of a “spacer molecule”. Such a spacer is often an aliphatic chain or a polyester structure similar to poly- or oligoethylene glycols. Preferably, the crosslinking agent is bi-, tri- or tetrafunctional, although bi- or tri-functional and bifunctional are most preferred. The inventors prefer to use bifunctional crosslinking linkers that readily interact under neutral to slightly alkaline conditions with high yields of interactions and in which the crosslinking molecule is consumed to a high degree. The inventors also prefer that the crosslinking molecules do not form by-products that must be removed before use. Many crosslinking agents are designed to cleave the leaving group upon reaction. In some cases, the inventors prefer crosslinking agents that exclude non-toxic components. Typical examples of such crosslinking functional groups are reactive esters, Michael acceptors, and epoxides. Preferred crosslinking molecules are ester derivatives of squaric acid, diepoxides and derivatives of acrylamides. Most preferred is diethyl squarate (3,4-diethoxy-3-cyclobutene-1,2-dione) and its structurally closely related analogues. Other preferred crosslinking linkers are 1,4-butanediol-diglycidyl ether, acrylamide derivatives and structurally closely related analogues thereof.

It is also important to minimize the amount of crosslinking reagent and to achieve an effective and pure coupling reaction, with the formation of a small amount, or without the formation of any by-products. The inventors prefer to use a low molar ratio of crosslinking agent to the number of functional amino groups in chitosan. The inventors prefer a molar ratio of 0.2: 1 or less, more preferably a ratio of 0.16: 1 or less, and most preferably a ratio of 0.1: 1 or less. The molar ratio is based on the number of groups available for crosslinking on the crosslinking linker and on chitosan. For a crosslinking linker, it will depend on functionality (bi-, tri-, tetrafunctional, etc.) and, on chitosan, on the availability of amino groups (only deacetylated amino groups will be reactive). Obviously, the amount of available amino groups will be determined by the degree of deacetylation of chitosan.

The inventors prefer that chitosan has a deacetylation degree below 75%, more preferably has a deacetylation degree below 70%, even more preferably has a deacetylation degree below 65% and even more preferably has a deacetylation degree of less than 60% and most preferably has a deacetylation degree of less than 55% . Chitin is completely insoluble in aqueous solutions and becomes somewhat soluble if the degree of deacetylation is 30% or more. The inventors prefer to have a degree of deacetylation above 35%, a degree of deacetylation above 40% is preferred, and a degree of deacetylation above 45% is most preferred. The solubility of chitosans also depends on parameters such as molecular weight, distribution of acetyl groups in the chain, and counterions. Chitosan is polydisperse in nature, that is, it contains a mixture of different chain lengths. On an industrial scale, chitosans are characterized by their viscosity, and give an average molecular weight. The inventors prefer to have a viscosity of up to 15,000 MPa · s, preferably from 2 to 10,000 MPa · s, more preferably from 5 to 2,000 MPa · s, and most preferably from 10 to 1,000 MPa · s when measured as 1% w / v . a solution in 1% aqueous acetic acid at a temperature of 25 ° C using a rotational viscometer with a rod rotating at 20 rpm The viscosity of a solution is an indicator of the average molecular weight of chitosan, while it is understood that chitosan is a polymer substance having a certain distribution of molecules with varying chain lengths. The inventors prefer to use chitosan solutions with a concentration of 3% or less. More preferably, a concentration of 2% or less is used. The inventors prefer to have a concentration above 0.3% (w / w).

The chitosan deacetylation pattern is also important for its properties. The chitosan of the present invention should be deacetylated randomly. That is, large blocks of a chitin-like polymer should be avoided, since such substances are prone to less solubility. Instead, the chitosan of the present invention has a random pattern of acetylated and deacetylated monosaccharide units. One way to determine the nature of monosaccharides is to determine the frequencies of nearest neighbors using NMR and to compare frequencies obtained using statistical models, see WO 03/011912.

Commercially available chitosan usually has a non-random block structure. The reason for this is that chitin is isolated in solid-state processes from shells of crustaceans. In such processes, when the shells remain undissolved throughout the process, the shells are treated with strong alkali to obtain partially deacetylated chitosan. However, since chitin is initially found in the form of a shell of a crustacean, hydroxide ions from alkali tend to affect mainly monosaccharide units on the surface of the shell; monosaccharide units in the center of a relatively thick shell usually do not encounter hydroxide ions and thus preserve the N-acetyl substitution pattern.

To avoid such chitin-like blocks, chitin / chitosan polysaccharide chains should be processed in solution. This allows the polysaccharide to enter the solution, and the shell structure is lost. This provides a random deacetylation scheme. It can be achieved by treating chitin in solution under strictly controlled conditions, or by completely deacetylating chitin and then reacetylating in solution to obtain the desired degree of deacetylation. See T. Sannan et al. Makromol. Chem. 177, 3589-3600, 1976, X. F. Guo et al. Journal of Carbohydrate Chemistry 2002, 21, 149-61 and K. M. Varum et al. Carbohydrate Polymers 25, 1994, 65-70. The chitosan of the present invention is preferably obtained by acetylation and / or deacetylation of chitosan in the solution phase to obtain a random deacetylation scheme.

If crosslinking of the low deacetylated chitosan is carried out, the inventors prefer to have interaction conditions where the pH is above 6 and where chitosan does not precipitate. It is even more preferable to use a pH above 6.5 and most preferably use a pH above 7.0. It is also preferable to use a pH that does not substantially destroy the crosslinking reagent by hydrolysis or by elimination reaction. Typical conditions for this reaction are alkaline conditions, and we prefer to use a pH below 10, more preferably a pH below 9.5, and even more preferably a pH below 9.0. Preferably, water is present in an amount of 97-99.7%. You can also use an additional solvent, such as ethanol, for example, in an amount of 0.2% (vol./vol.). The concentration of the crosslinking agent used in the crosslinking reactions is preferably 0.01-0.2% (vol./vol.), More specifically, above 0.02% (vol./vol.).

The viscoelastic hydrogel according to the invention is obtained in the form of a block, which can be isolated without further processing. The hydrogel is then processed to produce smaller blocks or fragments using conventional techniques known in the art. This resulting “crushed gel” is injected through a thin needle. The viscosity of the gel can be measured using a rheometer, as described in Example 16.

The present invention is further illustrated, but not limited, by the following examples.

Examples

Unless otherwise specified, the following substances were used in the Examples:

Chitosans with a low degree of N-deacetylation were obtained mainly following the principles set forth in: Sannan T, Kurita K, Iwakura Y. Studies on Chitin, 1. Die Makromolekulare Chemie 1975; 0: 1191-5, Sannan T, Kurita K, Iwakura Y. Studies on Chitin, 2. Makromol. Chem. 177, 3589-3600, 1976, Guo X, Kikuch, Matahira Y, Sakai K, Ogawa K. Water soluble chitin of low degree of deacetylation. Journal of Carbohydrate Chemistry 2002; 21: 149-61 and WO 03/011912.

Example 1

Chitosan (1.11 g, degree of N-deacetylation of 50%, MM (molecular weight) 145 kDa) was suspended in 70 ml of distilled water and 2 M HCl (aq) was added dropwise to dissolve chitosan. The pH of the solution was adjusted to 7.4 with 1M sodium hydroxide. The volume was adjusted to 100 ml with distilled water. 3,4-Diethoxy-3-cyclobutene-1,2-dione (122 μl of a 20% (v / v) solution in ethanol) was added, and the solution was stirred for 3 hours. The pH of the solution was adjusted to 8.3, and the volume was adjusted to 111 ml. The solution was placed in a thermostat at 40 ° C for 3 days. Hardened gel was designated as 1-1. The procedure was repeated, but 3,4-diethoxy-3-cyclobutene-1,2-dione was not added. This gel was designated as 1-2.

Example 2

Chitosan (0.50 g, degree of N-deacetylation 72%, MM 145 kDa) was suspended in 35 ml of distilled water, and 2 M HCl (aq) was added dropwise to dissolve chitosan. The pH of the solution was adjusted to 6.2 with 1 M sodium hydroxide. The volume was adjusted to 50 ml with distilled water. This gel was designated as 2-1. To 20 ml of the above solution was added 3,4-diethoxy-3-cyclobutene-1,2-dione (40 μl of a 12% (v / v) solution in ethanol), and the solution was stirred vigorously for 10 minutes. The pH of the solution was adjusted to 7.5. The solution was placed in a thermostat at 40 ° C for 3 days. The hardened gel was designated as 2-2.

Example 3

Chitosan (0.50 g, degree of N-deacetylation 72%, MM 145 kDa) crosslinked with glutaraldehyde (6 g) in 50 ml of 1 M HOAc (aq) at 100 ° C. Interaction conditions are as described in J. Control. Release 111 (2006), 281-289.

Example 4

1 g of each of the gels of Examples 1-4 was exposed to 1 M NaOH (aq.). Chitosan gels 1-2, 2-1 and 3 (comparative) were precipitated, while crosslinked gels 1-1 and 2-2 (invention) remained transparent.

Example 5

Chitosan hydrochloride (0.50 g, 55% N-deacetylation, MM 145 kDa) was dissolved in 45 ml of water. The pH of the solution was adjusted to 7.3 with dilute sodium hydroxide. 3,4-Diethoxy-3-cyclobutene-1,2-dione (102 μl of a 12% (v / v) solution in ethanol) was added, and the solution was stirred for 3 hours. The pH of the solution was adjusted to 8.3, and the volume was adjusted to 50 ml. The main feline allergen, Fel d 1 (5.9 mg) was added to 3 g of the above solution, and the mixture was transferred to a 5 ml vial and left for 6 days at 40 ° C. The resulting gel was mechanically treated and transferred into a 1 ml syringe.

Example 6

Hyaluronic acid (50 mg) was dissolved in MES (2- (N-morpholino) ethanesulfonic acid) buffer (20 ml, 20 mM, pH 6.5). The viscoelastic hydrogel from Example 5 (4 ml) was added to the hyaluronic acid solution and placed on the orbital shaker panel for 90 minutes. The coated gel was centrifuged at 2300 rpm for 2 × 10 minutes, washed with PBS buffer (phosphate-saline solution) and transferred to a syringe. This hyaluronic acid coated chitosan gel can be lyophilized and rehydrated to form a viscoelastic gel. When the hydrogel from Example 5 was added to the PBS buffer in the absence of hyaluronic acid, the thread-like fragments became whitish and sticky and could not be easily isolated, and therefore could not be rehydrated.

Example 7

Chitosan hydrochloride (0.30 g, 55% N-deacetylation, MM 145 kDa) was dissolved in 25 ml of water. The pH of the solution was adjusted to 8.3 with dilute sodium hydroxide, and the volume was adjusted to 3.0 g. A 3,4-diethoxy-3-cyclobutene-1,2-dione (61 μl of a 12% (v / v) solution was added to ethanol), and the solution was stirred for 3 hours, and then the mixture was left for 6 days at 40 ° C. 6 mg of Fel d 1 was added to this solution, and the mixture was stirred. Then the mixture was transferred into a 1 ml syringe.

Example 8 (comparative)

Chitosan hydrochloride (0.90 g, 81% N-deacetylation, MM 145 kDa) was suspended in 27 g of distilled water. The pH of the solution was 3.6. PBS (2.0 ml, 25 mM, pH 7.4) was added. The pH was adjusted to 5.8 with dilute sodium hydroxide and the volume was adjusted to 60 ml. 2.27 mg of Fel d 1 was mixed with 1.1 ml of the above solution and transferred into a 1 ml syringe.

Example 9

Groups of BALB / c mice were injected subcutaneously, in the neck, with 100 μl of the solutions of Examples 5, 7 and 8. Then, the mice were killed at different time points, after 1, 7 and 21 days. Mice were killed by inhalation of CO ₂ . The skin at the injection site was collected and placed in Histocon on ice, and then skin samples were frozen in an acetone bath. Frozen biopsy skin samples were stored at -80 ° C until histological section. Then the histological sections were analyzed for cell infiltration. Histological examination showed extensive cell infiltration already on the first day after injection for the gels of Examples 5 and 7. The histological examination of the gel of Example 8 showed a biconvex gel with a surface covered by cells, but without cells inside the gel. In gels 5 and 7, the cells were infiltrated into the whole substance after 24 hours. After 7 days, the amount of chitosan decreased in the gels of Examples 5 and 7. Two weeks later, on day 21, there was virtually no trace of the injected substance, while the gel from Example 8 showed much slower cell colonization and also much slower decomposition.

Figure 1 shows the histological sections of the injection sites of mice 24 hours after subcutaneous injection with the viscoelastic chitosan gel from Example 5 (Figure 1 (a)) and the comparative chitosan from Example 8 (Figure 1 (b)), respectively. The results indicate that the gel of the present invention is infiltrated by immune cells faster and to a greater extent than the comparative gel. Even after three weeks, the comparative chitosan had not yet been completely infiltrated, and a slower decomposition rate was observed for the comparative chitosan gel compared to the gel of the invention.

Example 10

Groups of three BALB / c mice were injected subcutaneously, in the neck, with 100 μl of the gel of Examples 5, 7, and 8. Booster injection was performed on day 64, 9 weeks after the first injection. Then, blood samples were taken from the caudal artery at 1, 2, 3, 9, and 10 weeks after injection. The levels of IgG ₁ and IgE specific serum to Fel d 1 were measured using ELISA (ELISA). All gels elicited an IgG ₁ antibody response.

Example 11

Chitosan (2.05 g, degree of N-deacetylation of 50%, MM 145 kDa) was suspended in 160 ml of distilled water and 2 M HCl (aq) was added until the chitosan was dissolved. The pH was adjusted to 7.9 by dropwise adding 1 M NaOH (aq). The volume was adjusted to 200 ml with distilled water. 1,4-butanediol diglycidyl ether (166 μl of a 5% (v / v) solution in ethanol) was added dropwise to 50 ml of the above solution. The mixture was vigorously stirred for 10 minutes at room temperature and then placed in a thermostat (50 ° C) overnight.

Example 12

Chitosan (2.25 g, 55% N-deacetylation degree, MM 145 kDa) was suspended in 130 ml of distilled water, and 2 M aqueous hydrochloric acid was added until the chitosan was dissolved. The pH was adjusted to 6.75 with 1 M sodium hydroxide, and the volume was adjusted to 160 ml. To 50 ml of the above solution was added 3,4-diethoxy-3-cyclobutene-1,2-dione (120 μl, 12% solution in ethanol), and the solution was stirred for 2 hours at room temperature. Diclofenac (773 mg) was dissolved in 25 ml of distilled water and added to the above solution. The pH of the solution was adjusted to 8.1 with 1 M sodium hydroxide, and the solution was sonicated for 1 h and then heated at 40 ° C. overnight. The resulting gel (1 g) was mixed with hyaluronic acid (4 g, 0.25% in distilled water). The gel was placed in a Franz cell equipped with a Spectra / For filter with a molecular weight cut-off threshold of 2000 Da and filled with PBS buffer. 38% of diclofenac was released after 2 hours, 60% after 5 hours and 72% after 24 hours

Example 13

The procedure of Example 5 was repeated with rFel d 1 radiolabeled with ⁷⁵ Se. The preparation of rFel d 1 labeled with ⁷⁵ Se was performed using in situ labeling of the selenocysteine residue in the Sel-labeled rFel d 1, essentially as described previously for Der p 2 (Febs J 2005; 272: 3449-60), but using the constructs , Preparation and Purification Conditions for Sel-Labeled rFel d 1 (Chembiochem 2006; 7: 1976-81).

Example 14

In vivo tracking of 100 μg of radiolabeled [ ⁷⁵ Se] rFel d 1 (2 μKu) bound to chitosan or adsorbed onto aluminum hydroxide was performed as previously described (Febs J 2005; 272: 3449-60, Methods Enzymol 1981; 77: 64 -80). Briefly, mice (n = 2 / group) were injected subcutaneously with Chitosan- [ ⁷⁵ Se] rFel d 1 or aluminum alum- [ ⁷⁵ Se] rFel d 1 and were killed after 24 hours or 1 week. The mice were frozen and subjected to a tape sectional autoradiography process. Sections (60 μm) were pressed onto an X-ray film (Structurix, Agfa, Mortsel, Belgium) and developed using D19 (Kodak, Rochester, USA).

Results: After 24 hours, radioactivity was metabolized and determined, for example, in the liver and spleen. The picture was like aluminum hydroxide. After 1 and 2 weeks, respectively, only trace amounts of radioactivity could be detected.

Example 15

Chitosan (3.6 g, 52% N-deacetylation) was suspended in 250 ml of distilled water and 2 M HCl (aq) was added until the chitosan was dissolved. The pH was adjusted to 7.0 by dropwise adding 1 M NaOH (aq). The volume was adjusted to 300 ml with distilled water. This solution was designated as solution X (1.2% chitosan). 50 ml of water was added to 100 ml of solution X, this solution was designated as solution Y (0.8% chitosan). 16 ml of solution X and solution Y were respectively added to two beakers for each solution. Different amounts of 3,4-diethoxy-3-cyclobutene-1,2-dione (10% (v / v) solution in ethanol) were added to each according to the list below.

X-1 20 μl

X-2 59 μl

X-3 118 μl

Y-1 29 μl

Y-2 88 μl

Y-3 176 μl

The mixture was intensively stirred for 10 minutes at room temperature, and then 4 g of each solution was transferred to Petri dishes (d = 35 mm), hermetically closed and placed in a thermostat (40 ° C) for 4 days.

Cylinders with a diameter of 6 mm and a height of 2.65 ± 0.55 mm were removed from the Petri dishes and the gel disks were compressed using an Instron 3345 equipped with a 100 N load cell. The samples were compressed at 1 mm / min.

It should be mentioned that gels based on a 0.8% chitosan solution (Y-1, Y-2, Y-3) were technically more difficult to handle due to their less rigid structure, and thus analytical accuracy was reduced compared to with gels based on a 1.2% solution of chitosan (X-1, X-2, X-3). Although this should be taken into account when comparing analytical data, the gel was not adversely affected (a really less rigid structure can make the gel more malleable for crushing). Analytical data based on measurements of 1.2% gels (X-1, X-2, X-3) showed that the average value of the modulus E increased from 4.7 to 14.1 MPa, when the amount of cross-linking agent increased from 2 to 12 % (calculated as the ratio between crosslinking agent and monosaccharide units).

Example 16

Chitosan (4 g, 55% N-deacetylation) was suspended in 350 ml of distilled water, and 2 M HCl (aq) was added until the chitosan was dissolved. The pH was adjusted to 7.0 by dropwise adding 1 M NaOH (aq). The volume was adjusted to 400 ml with distilled water. Two different volumes of 3,4-diethoxy-3-cyclobutene-1,2-dione (60 (sample 1) and 185 μl (sample 2) 12% (v / v) solution in ethanol) were added dropwise to two different measuring cups containing 50 ml of chitosan solution. The mixture was vigorously stirred for 5 minutes at room temperature and then 8 ml was transferred into plastic syringes (10 ml). The syringes were hermetically closed and placed in a thermostat (40 ° C) for 72 hours. Then, the formed gels were transferred into a new syringe (5 ml syringes), squeezing them through a silicone tube (d = 3 mm). Syringes were stored at 4 ° C before measurement. For rheological studies, the Bohlin Gemini VOR instrument was used, using a geometry with a cone and plate with a diameter of 40 mm and a cone angle of 4 degrees for the measuring cell. All measurements were performed at 25 ° C.

Storage and loss factors G 'and G' 'were investigated in pulsating shear experiments. The rheological parameters reflected the viscoelastic properties of a solid and a liquid, respectively.

Both gel samples showed the properties of a viscoelastic plastic solid in measuring the stress range with G '> G' ', i.e. the elastic component is larger than the liquid component. Within a stable linear region, G '(1 Hz) was approximately 450 Pa with a phase angle of approximately 1 ° for sample 1. For sample 2, the corresponding data was G' of approximately 900 Pa and a phase angle of 1 °.

The second type of oscillation measurements performed on the same preparations were frequency swings for a constant strain of 0.5. The following observations were made: the gel of sample 2 has a higher gel strength with an increased elastic modulus G 'than the gel of sample 1, and both samples show the apparent frequency of the independent elastic modulus in the studied range of 0.1-20 Hz.

It is possible to perform reproducible measurements of viscoelastic properties on fragmented versions of gel samples. Two gel samples exhibit substantially the same viscoelastic properties in a fragmented state. The gel sample 2 has a greater gel strength compared to sample 1.

Example 17

Chitosan (1 g, 55% N-deacetylation degree) was suspended in 80 ml of distilled water, and 2 M HCl (aq) was added until the chitosan was dissolved. The pH was adjusted to 6.8 by dropwise adding 1 M NaOH (aq). The volume was adjusted to 100 ml with distilled water. PEGl 600 (2.5 g, 40% dissolved in water) and a 3,4-diethoxy-3-cyclobutene-1,2-dione (183 μl of a 1% (v / v) solution in ethanol) were added dropwise at stirring to 7.5 g of chitosan solution. The solution was placed in a thermostat at 40 ° C for 3 days to obtain a transparent viscoelastic gel.

Example 18

Chitosan (1.5 g, 55% N-deacetylation) was suspended in 80 ml of distilled water and 2 M HCl (aq) was added until the chitosan was dissolved. The pH was adjusted to 6.5 by dropwise adding 1 M NaOH (aq). The volume was adjusted to 100 ml with distilled water. Metagin (0.2 g) and propagin (0.03 g) dissolved in 23 g of water were added to 67 g of chitosan solution and stirred for 18 hours at room temperature. 3,4-Diethoxy-3-cyclobutene-1,2-dione (24.6 μl of an 11% (v / v) solution in ethanol) was added dropwise to the above solution. The solution was placed in a thermostat, as described in Example 17.

Claims (12)

1. A crosslinkable chitosan composition containing chitosan with a degree of deacetylation of 30-75%, where chitosan is deacetylated randomly, and a crosslinking agent, where the molar ratio of crosslinking agent to chitosan is 0.2: 1 or less, calculated on the number of functional groups in the crosslinking agent and the number of deacetylated amino groups in chitosan. 2. A crosslinkable chitosan composition according to claim 1, wherein the chitosan has a deacetylation degree of 35-55%. 3. The crosslinkable chitosan composition according to claim 1, wherein the chitosan prior to crosslinking has an average molecular weight of 10-500 kDa. 4. A crosslinkable chitosan composition according to claim 1, wherein the crosslinking agent is bifunctional. 5. A crosslinkable chitosan composition according to any one of claims 1 to 4, wherein the crosslinking agent has functional groups selected from esters, Michael acceptors, epoxides, and combinations thereof. 6. A method of producing a chitosan hydrogel, comprising obtaining a crosslinkable chitosan composition according to any one of claims 1 to 5 in an aqueous solution, crosslinking the composition and isolating the obtained chitosan hydrogel. 7. The method according to claim 6, where the crosslinking is performed at a pH of 6-10. 8. Chitosan hydrogel obtained by the method according to claim 6 or 7. 9. The chitosan hydrogel of claim 8 in the form of a crushed gel. 10. The chitosan hydrogel of claim 8 or 9 for use as a vaccine, in drug delivery, in tissue extension, as a scaffold of cell cultures, for encapsulating viable cells, in wound healing devices, in orthopedics, as biomaterial, treatment of urinary incontinence or vesicourethral reflux, in viscosurgery, in the provision of living cells to the host body, as a cosmetic, as a volume-giving agent, as a thickener, as an additive in the food industry as an adhesive, as a lubricant, or as an auxiliary fluid for drilling. 11. A pharmaceutical composition for delivering an active agent comprising a chitosan hydrogel according to claim 8 or 9 and a pharmaceutically active ingredient. 12. An immunological agent containing a chitosan hydrogel according to claim 8 or 9 as an adjuvant and an antigen, where the antigen is possibly covalently linked to chitosan.

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